We are analyzing the interactions between virus glycoproteins and their receptors for several enveloped viruses of public health significance in the US and worldwide. Our goals are to define mechanisms of virus entry and cell tropism, identify cellular receptors that mediate virus entry, and develop novel antiviral approaches for treatment (antiviral drugs) and prevention (vaccines, microbicides). 1) Kapsosi's Sarcoma-Associated Virus (KSHV, aka human herpesvirus 8). Our previous work demonstrated that a broad range of human and non-human primate cell types can serve as efficient targets in assays of KSHV glycoprotein-mediated cell fusion and KSHV virion entry. With this information, we devised a strategy for KSHV receptor identification involving functional selection of a cDNA library derived from a highly permissive target cell. The receptor discovered by this method is xCT, the 12 transmembrane light chain of the xc- cystine/glutamate exchange transport system. During the past year, we obtained structure/function insights into the mechanism of KSHV glycoprotein interaction with xCT. Our results indicate that different subsets of glycoproteins interact independently with xCT to promote fusion, apparently at different sites on the receptor. We have designed soluble constructs of specific KSHV glycoproteins in order to directly analyze their binding to xCT. We have also initiated studies of KSHV entry into B cells, which are major viral reservoirs in infected people. We have planned collaborative studies (C. Rinaldo, U. Pittsburgh) to assess the role of xCT in KSHV entry into primary B cells, and to elucidate the regulation of this and other candidate receptor molecules at various stages of B cell maturation. 2) Hepatitis C virus (HCV). We are particularly interested in developing systems to study HCV entry, for use in defining essential receptors and elucidating entry mechanisms, assaying HCV neutralizing antibodies in infected people and in vaccine studies. To this end, we are devising systems to produce HCV virus-like particles containing the functional HCV E1 and E2 glycoproteins and containing an RNA that encodes a reporter gene; this will enable rapid, quantitative, and sensitive measurement of entry. The first approach employs a recombinant baculovirus system, which to date is the only approach that has yielded large amounts of purified particles composed of the HCV structural genes (core, E1, E2). We have devised several approaches to encapsidate the desired reporter gene-encoding RNA. In the second approach, we are seeking to develop chimeric VLPs containing the core of West Nile virus, an ecapsidated reporter RNA, and HCV glycoproteins on the surface. We have obtained preliminary evidence that under certain conditions, HCV E1 and E2 can be incorporated into such chimeric particles, and can facilitate entry by virtue of E2 binding to CD81 on the target cell surface.